Study of intracellular calcium movement by time resolved image processing method.

众所周知，肌肉收缩是由细胞内钙离子浓度增加引起的。该项目的目的是使用视频增强显微镜系统和图像处理器来观察单个骨骼肌纤维中的钙浓度是否均匀增加。以生物发光蛋白Aequorin作为钙指示剂，同时记录光电倍增管信号和Aequorin发光图像。1.向单个骨骼肌纤维内注射水飞蓟素，诱发抽动。在注射赤藜芦素的区域观察到了发光点。如果加上几个抽动的图像，在Aequorin注射区域观察到的亮度没有实质性的差异。2.在强直的骨骼肌纤维(40 Hz，1s)中，在Aequorin注射区域添加多幅图像后，张力保持不变，亮度没有明显差异。当肌节从2.4微米改变为3.6微米时，也得到了类似的结果。已知从肌浆网释放钙的咖啡因导致肌挛缩，出现在制剂表面的光点扩散到纤维的中心。最后，水飞蓟素载药区变得均匀明亮。这些结果表明，在显微镜和视频分辨率的限制下，生理激活不会引起细胞内钙离子浓度的不均匀，而咖啡因扩散到纤维中会导致钙离子释放的不均匀。引起不均匀活化的条件及其意义尚需进一步研究

Muscle contraction is known to be triggered by an increase of intracellular calcium ion concentration. The aim of the project was to observe whether calcium concentration increases homogeneously in a single skeleral muscle fiber using video-intensified microscopy system and image processor. Bioluminescent photoprotein, aequorin, was used as a calcium indicator, and phtomultiplier signal and image of aequorin luminescence were simultaneously recorded. 1. Aequorin was injected into a single skeletal muscle fiber and twitch was initiated. Luminescent spots were observed in the area where aequorin was injected. If several images of twitches were added, no substantial difference of brightness was observed in the aequorin injected area. 2. In tetanized skeletal muscle fibers (40 Hz, 1 sec), tension was sustained and no significant difference of brightness was recognized after adding several images in the aequorin injected area. Similar result was obtained when the sarcomere was changed form 2.4<micrn>m to 3.6<micrn>m. Caffeine known to release calcium from sarcoplasmic reticulum caused contracture, and luminescent spots which appeared in the surface of the preparation spreaded into the center of the fiber. Finally, the aequorin loaded area became bright homogeneously. These results indicate that physiological activation dose not cause inhomogeneity of intracellular calcium concentration under the limitation of microscopic and video resolution and that caffeine which diffuses into the fiber causes inhomogeneous calcium ion release. Further study is required to clarify the condition which causes inhomogeneous activation and its significance